Levofloxacin veterinary formulations are available in Argentina, China and India for the use in dogs, cattle, pig and sheep, but not currently in the rabbit. Only the extra‐label use in rabbits is possible. Levofloxacin is not labelled for veterinary use in the EU or the USA. The activity of levofloxacin against rabbit pathogens Pasteurella multocida (P. multocida) and Escherichia coli (E. coli) was evaluated. Minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined in broth and serum for 10 P. multocida isolates and 5 E. coli isolates from rabbits. One isolate of each bacterial species was used for the time‐killing curve study in vitro and ex vivo. In vitro AUC24/MIC ratios were used for building the inhibitory pharmacodynamic Imax model. The P. multocida MIC were 0.008–0.5 μg/mL, MBC – 0.015–0.5 μg/mL. Escherichia coli MIC was 0.008–0.03 μg/mL and MBC – 0.03–0.25 μg/mL. Bacterial counts were reduced to the limit of detection after 24 h with levofloxacin concentrations of 2 MIC and higher. All serum samples from rabbits treated with levofloxacin eliminated the bacteria within 24 h. AUC24/MIC ratios for bacteriostatic, bactericidal and bacterial elimination effects for P. multocida and E. coli isolates were 21, 29 and 75 h and 27, 32 and 60 h, respectively. Proposed daily doses against P. multocida (MIC = 0.015 μg/mL) and E. coli (MIC = 0.03 μg/mL) isolates were calculated as ≤0.91 and ≤1.43 mg/kg, respectively. Fluoroquinolones are categorized by WHO as ‘highest priority critically important antimicrobials’. Considering the increasing importance of antimicrobial stewardship, antimicrobials from a lower importance category that are active against the isolate of interest should be used in preference to fluoroquinolones. Fluoroquinolone use in veterinary medicine should be based on antimicrobial susceptibility testing in order to mitigate the risk to public health and prevent the spread of bacterial resistance.